Soil nitrification inhibitor and application thereof

Abstract

The invention discloses a soil nitrification inhibitor and application thereof, and belongs to the field of environmental protection and nitrification inhibitors, the soil nitrification inhibitor is vanillic acid, can effectively inhibit the nitrification effect of rice and wheat rotation soil and reduce farmland nitrogen loss and soil N2O emission, and the inhibition efficiency is superior to that of other nitrification inhibitors. The vanillic acid is stable in chemical structure, belongs to a'green 'biological nitrification inhibitor of a plant source, and can solve the problems that an existing nitrification inhibitor is high in price, unstable in effect and prone to affecting the environment and food safety.

Description

Technical Field

[0001] The present invention belongs to the fields of environmental protection and nitrification inhibitors, and particularly relates to a soil nitrification inhibitor and its application. Background Art

[0002] As a key production factor to ensure crop yield increase and food security, the scientific application of fertilizers is crucial. The situation of excessive fertilization will stimulate the nitrification reaction driven by microorganisms in the soil, resulting in nitrogen loss. According to relevant research estimates, among the total nitrogen losses, the losses caused by the nitrification process account for up to 40%. At the same time, with the continuous increase in the amount of nitrogen fertilizer input, the emissions of N2O (nitrous oxide) will also increase exponentially. In view of this, regulating the nitrification process in the soil to reduce nitrogen loss is of great significance for promoting the sustainable development of global agriculture, ensuring food security, and effectively responding to climate change.

[0003] Currently, applying nitrification inhibitors in agricultural production is an important measure to improve nitrogen use efficiency and reduce nitrogen loss. Nitrification inhibitors inhibit the activity of related soil microorganisms, slow down nitrification, so that more nitrogen is retained in the soil in the form of ammonium nitrogen (NH4 + -N) for crop absorption and utilization, realizing efficient management of nitrogen fertilizers and reducing the negative effects caused by excessive fertilization. However, most of the currently used nitrification inhibitors are prepared by chemical synthesis, which have limitations such as high price, unstable effects, and potential threats to environmental and food safety, and are difficult to meet the requirements of green agriculture standards. For example, the nitrification inhibitor dicyandiamide (DCD) can only effectively play an inhibitory role when the application amount is relatively high. Since it is easily soluble in water, it is easy to leach; 2-chloro-6-(trichloromethyl)pyridine (Nitrapyrin) is a chlorine-containing organic compound, and long-term application will have an adverse impact on the environment, and it is volatile and photodegradable, and has certain corrosiveness and explosiveness and other problems. Therefore, there is an urgent need in this field to seek an environmentally friendly alternative solution for controlling nitrification.

[0004] As a new solution, plant-derived biological nitrification inhibitors can avoid the ecological risks of chemical agents due to their natural characteristics. They are more environmentally friendly than synthetic nitrification inhibitors and meet the needs of the green development of agriculture. The nitrification-inhibiting substances in the root exudates of crops reported in the existing literature, such as methyl p-hydroxybenzoate, 1,9-decanediol, etc., although they have good nitrification inhibition effects, currently have high synthesis costs and are limited in popularization and application. Therefore, it is very necessary to continue to explore the substances in crop root exudates that have the effect of inhibiting soil nitrification. Summary of the Invention

[0005] In view of the above problems, the present invention provides a soil nitrification inhibitor and its application to solve the problems of high price, short aging time, and easy impact on environmental and food safety of existing chemically synthesized nitrification inhibitors, and further expand the application fields of vanillic acid.

[0006] One of the technical solutions provided by the present invention:

[0007] Use of vanillic acid in inhibiting soil nitrification.

[0008] Another technical solution provided by the present invention:

[0009] A soil nitrification inhibitor, wherein the soil nitrification inhibitor is vanillic acid.

[0010] Another technical solution provided by the present invention:

[0011] An application of the above soil nitrification inhibitor in inhibiting soil nitrification during crop planting, wherein vanillic acid and nitrogen fertilizer are formulated into a mixed solution and applied to the soil.

[0012] The mechanism of vanillic acid in inhibiting soil nitrification in rice-wheat rotation includes:

[0013] (1) As a natural organic acid, the acidic substances released during the decomposition of vanillic acid will reduce the soil pH value. Nitrification is stronger under alkaline conditions, and when the pH decreases, the nitrification rate drops significantly. The presence of vanillic acid can make the soil tend to an acidic environment, directly inhibiting the activity of nitrifying bacteria; (2) Vanillic acid itself has antibacterial properties and may directly interfere with the enzyme system or metabolic pathway of nitrifying bacteria; (3) Vanillic acid can increase soil organic matter. After the organic matter content increases, the activity of heterotrophic microorganisms increases, competing with nitrifying bacteria for oxygen and nutrient resources, which can indirectly inhibit the nitrification process.

[0014] Furthermore, 100 - 2000 mg of the vanillic acid is applied per kilogram of soil; the application rate of the nitrogen fertilizer is calculated as 100 mg N per kilogram of soil.

[0015] Even further, 100 - 500 mg of the vanillic acid is applied per kilogram of soil.

[0016] Even further, 100 mg of the vanillic acid is applied per kilogram of soil.

[0017] Furthermore, the crop planting is rice-wheat rotation.

[0018] In the rice-wheat rotation system, nitrification is a key process of soil nitrogen transformation. However, excessive nitrification can lead to nitrogen loss in the form of nitrate and reduce the nitrogen fertilizer utilization rate. As a phenolic acid substance, vanillic acid may inhibit nitrification through the following mechanisms: First, directly inhibiting the activity of nitrifying microorganisms. Vanillic acid may have a toxic effect on nitrifying microorganisms such as nitrite bacteria and nitrate bacteria, hindering the process of converting ammonium nitrogen into nitrate nitrogen. Second, changing the soil microbial community structure. Vanillic acid may indirectly affect nitrification by selectively inhibiting or promoting certain microbial populations and changing the soil microbial community structure. Third, affecting the soil environment. Vanillic acid may create conditions unfavorable for the growth of nitrifying microorganisms by changing environmental factors such as soil pH value and redox potential.

[0019] Furthermore, the physical and chemical properties of the soil are as follows: the soil-water mass ratio is calculated as 2.5∶1, the pH is 8.54, the organic matter content is 17.37 g / kg, the total nitrogen content is 0.89 g / kg, the available nitrogen content is 69.81 mg / kg, the available phosphorus content is 7.16 mg / kg, and the available potassium content is 124.5 mg / kg.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The vanillic acid of the present invention can effectively inhibit nitrification in the rice-wheat rotation soil. At a relatively low dose (100 mg / kg soil), it can reduce farmland nitrogen loss and soil N2O emissions, and its inhibition efficiency is better than that of other nitrification inhibitors. Vanillic acid has a stable chemical structure and belongs to a plant-derived "green" biological nitrification inhibitor, which can solve the problems of high price, unstable effect, and easy impact on environmental and food safety of existing nitrification inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is a relative abundance analysis diagram of the identifiable components of pumpkin root exudates;

[0024] Figure 2 It is a relationship diagram between the application amount of vanillic acid and the nitrification inhibition effect in Example 1;

[0025] Figure 3 It is the influence of the application amount of vanillic acid on soil N2O emissions in Example 2;

[0026] Figure 4 Graph for comparing the nitrification inhibition capabilities of vanillic acid, methyl 4-hydroxycinnamate (MHPP), and dicyandiamide (DCD) in Example 3;

[0027] Figure 5 Graph for comparing the abundances of ammonia-oxidizing archaea (AOA) under the conditions of applying vanillic acid, methyl 4-hydroxycinnamate (MHPP), and dicyandiamide (DCD) in Example 4;

[0028] Figure 6 Graph for comparing the abundances of ammonia-oxidizing bacteria (AOB) under the conditions of applying vanillic acid, methyl 4-hydroxycinnamate (MHPP), and dicyandiamide (DCD) in Example 4. Detailed implementation manners

[0029] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0034] The room temperature in the present invention refers to 25 ± 2 °C.

[0035] Vanillic acid (VA) is an organic compound with the chemical name 4-hydroxy-3-methoxybenzoic acid, a molecular formula of C8H8O4, and a molecular weight of 168.15. Vanillic acid widely exists in nature, such as in many plants and essential oils like vanilla beans, vanilla pods, Peru balsam, benzoin balsam, and Javanese perfume oil. It is also one of the metabolites of tyrosine and catecholamines. Vanillic acid is used as a flavoring agent in the food industry and is widely applied in the food industry. It is used to make spices and flavorings due to its hepatoprotective effect and antioxidant properties. Vanillic acid can inhibit the activation of NF-κB and has anti-inflammatory activity. By acting on the immune-mediated aspect of concanavalin A-induced liver injury, it has an inhibitory effect on liver cell inflammation. In vitro studies have shown that vanillic acid can significantly reduce the ROS level, indicating its effective antioxidant capacity. Therefore, it is also widely used in the pharmaceutical and skincare fields, but its application in the fields of environmental protection and nitrification inhibitors has rarely been reported.

[0036] The present invention further expands the application fields of vanillic acid, specifically including: using it to prepare soil nitrification inhibitors to regulate the soil nitrification process; developing related products to reduce nitrogen loss in farmland and improve nitrogen fertilizer utilization rate to contribute to the sustainable development of agriculture; reducing the emission of N2O in the soil through its mechanism of action to alleviate the greenhouse effect; at the same time, using the inhibitory effect of vanillic acid on nitrifying microorganisms (including ammonia-oxidizing archaea and ammonia-oxidizing bacteria) to regulate their activity and abundance, providing a new way for the ecological regulation of soil microorganisms.

[0037] The root exudates of pumpkins in the present invention were collected and concentrated, and their components were identified by gas chromatography-mass spectrometry (GC / MS). The identification results are as Figure 1 shown, indicating that vanillic acid exists in the root exudates and is a biological inhibitor derived from plants.

[0038] In the examples of the present invention, the tested soil was collected from the plow layer soil (0 - 20 cm) of a rice-wheat rotation experimental field in Huaiyin District, Huai'an City, Jiangsu Province, and the soil type is yellow brown soil. The basic physical and chemical properties of the soil are: pH is 8.54 (soil-water ratio 2.5∶1, w / w), organic matter content is 17.37 g / kg, total nitrogen content is 0.89 g / kg, available nitrogen content is 69.81 mg / kg, available phosphorus content is 7.16 mg / kg, and available potassium content is 124.5 mg / kg. The tested products vanillic acid, dicyandiamide, and methyl 4-hydroxybenzoate were all obtained through commercial purchase. The experiment adopted the indoor soil culture method, and all were cultured in 100 mL glass bottles. The tested nitrogen fertilizer was (NH4)2SO4 (N 100 mg / kg soil).

[0039] Example 1: Effects of adding different concentrations of vanillic acid on soil nitrification potential

[0040] Set up soil samples in the ammonium sulfate treatment group (CK) and soil samples in the ammonium sulfate + vanillic acid treatment group. The application rate of ammonium sulfate is 100 mg N / kg, and vanillic acid has 4 addition amounts, which are 100, 500, 1000, and 2000 mg / kg soil respectively, numbered VA-100, VA-500, VA-1000, and VA-2000. Each of the above treatments has 3 replicates.

[0041] Calculate the nitrification potential of the soil: Weigh 5.0 g of the above soil samples respectively and place them in 50 mL centrifuge tubes. Add 20 M phosphate buffer solution (8.0 g / L NaCl, 0.2 g / L KCl, 0.2 g / L Na2HPO4, pH 7.1), and add 1 mmol / L (NH4)2SO4 and 50 mg / L KClO3 to inhibit nitrite oxidation. Place the centrifuge tubes at room temperature and shake and culture them at a speed of 150 revolutions per minute for 24 hours. After the culture is completed, add 5 mL of potassium chloride (KCl) solution with a concentration of 2 mol / L to the centrifuge tubes to extract nitrite nitrogen (NO2--N) in the soil. Subsequently, use 1-naphthylamine and sulfanilic acid as chromogenic reagents to measure the concentration of NO2--N in the extract, and perform colorimetric analysis on the microplate reader at a wavelength of 530 nm. Calculate the nitrification potential of the soil according to the increase in the content of NO2--N in the unit soil sample per unit time.

[0042] The experimental results are as Figure 2 shown in the relationship diagram between the application amount of vanillic acid and the nitrification inhibition effect. Compared with the control without adding vanillic acid, the inhibition rates of vanillic acid with addition concentrations of 100, 500, 1000, and 2000 mg / kg soil on the nitrification potential are 83.9%, 90.8%, 94.2%, and 96.0% respectively.

[0043] Example 2: Effects of adding different concentrations of vanillic acid on soil N2O emissions

[0044] Weigh 5 portions of 10 g of fresh soil and place them in 100 mL culture flasks respectively. Add distilled water to adjust the soil water content to 40% of the field water holding capacity (WHC), and incubate at a constant temperature of 25 °C in the dark. Set up an ammonium sulfate treatment group (CK) and an ammonium sulfate + vanillic acid treatment group: Weigh 4 different masses of vanillic acid according to the experimental treatment and add them to (NH4)2SO4. Add 4 of them to the soil and mix well. The application rate of (NH4)2SO4 in the prepared soil samples is 100 mg N / kg, and the application rates of vanillic acid are 100, 500, 1000, and 2000 mg / kg soil respectively, numbered VA-100, VA-500, VA-1000, and VA-2000. Add ammonium sulfate to the remaining portion of fresh soil, with an application rate of 100 mg N / kg, as the CK group. Adjust the water content of the above five soil samples to 60% WHC. Every 3 days, open the bottles for aeration and weigh them, and then add an appropriate amount of deionized water to maintain the soil moisture. Collect N2O gas on the 1st, 2nd, 3rd, 4th, 5th, 7th, and 14th days after adding the treatment solution. One day before each sampling, exhaust the glass bottles and seal them with rubber stoppers for 23 h. When sampling, use a 25 mL syringe to extract repeatedly 3 times and then collect 20 mL of gas and inject it into a vacuum bottle for measuring the N2O concentration.

[0045] The experimental results are as Figure 3 shown in the effect of vanillic acid application rate on soil N2O emissions. Vanillic acid significantly inhibited soil N2O emissions from the first day in paddy soil. Different dosage treatments all delayed the N2O emission peak, and with the increase of dosage, the inhibitory effect gradually enhanced.

[0046] Example 3 Effect of different nitrification inhibitors on soil nitrification potential

[0047] Set up an ammonium sulfate treatment (CK) and an ammonium sulfate + different nitrification inhibitor treatment. The application rate of ammonium sulfate is 100 mg N / kg. Take 3 kinds of nitrification inhibitors, namely vanillic acid (VA), methyl 4-hydroxybenzenepropionate (MHPP), and dicyandiamide (DCD), numbered VA-100, MHPP, and DCD respectively. The application rates of VA, MHPP, and DCD are all 100 mg / kg soil, and each treatment is set with 3 replicates.

[0048] The experimental steps for calculating the nitrification potential of the soil are the same as those in Example 1. The experimental results are as Figure 4 shown in the comparison chart of the nitrification inhibition ability of vanillic acid, methyl 4-hydroxybenzenepropionate (MHPP), and dicyandiamide (DCD). Among the 3 nitrification inhibitors, the nitrification potential of the treatment with vanillic acid added is the lowest. After calculation, the inhibition efficiency of vanillic acid on nitrification potential is 83.9%, the inhibition efficiency of methyl 4-hydroxybenzenepropionate on nitrification potential is 65.5%, and the inhibition efficiency of dicyandiamide on nitrification potential is 80.0%. It shows that the inhibition efficiency of vanillic acid on nitrification potential is higher than that of methyl 4-hydroxybenzenepropionate and dicyandiamide.

[0049] Example 4. Effects of Different Nitrification Inhibitors on the Abundance of Soil Nitrosifying Bacteria

[0050] Prepare mixed solutions of different nitrification inhibitors (vanillic acid, methyl 4-hydroxybenzenepropionate, and dicyandiamide) and ammonium sulfate. Weigh 12 portions of 10 g of fresh soil into 100 mL culture flasks, and evenly add 2 mL of the mixed solution prepared from each nitrification inhibitor and ammonium sulfate to the culture flasks to obtain different treatment groups, including vanillic acid + ammonium sulfate (numbered VA-100, application rate: VA is 100 mg / kg, ammonium sulfate is 100 mg N / kg), methyl 4-hydroxybenzenepropionate + ammonium sulfate (numbered MHPP, application rate: MHPP is 100 mg / kg, ammonium sulfate is 100 mg N / kg), dicyandiamide + ammonium sulfate and ammonium sulfate (numbered DCD, application rate: DCD is 100 mg / kg, ammonium sulfate is 100 mg N / kg), and a blank group (CK, application rate: ammonium sulfate is 100 mg N / kg). Each treatment is set with 3 replicates. Place the culture flasks in a constant temperature (room temperature) incubator for light-shielded culture for 7 days. After 7 days, collect the soil samples in the culture flasks, extract the soil DNA samples, and quantitatively detect the abundances of nitrosifying bacteria in the samples, mainly including ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB).

[0051] Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), as the core functional microbial groups in the soil ammonia oxidation process, their community abundances, etc. are the key biological factors regulating the intensity of soil nitrification. The detection results of the abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the samples are respectively as Figure 5 and Figure 6 shown. From Figure 5 the comparison chart of the abundances of ammonia-oxidizing archaea (AOA) under the application conditions of vanillic acid, methyl 4-hydroxybenzenepropionate (MHPP), and dicyandiamide (DCD), it can be seen that the AOA abundance in the treatment with vanillic acid is similar to that in the MHPP treatment, and both are significantly lower than the control and DCD treatments. From Figure 6 the comparison chart of the abundances of ammonia-oxidizing bacteria (AOB) under the application conditions of vanillic acid, methyl 4-hydroxybenzenepropionate (MHPP), and dicyandiamide (DCD), it can be seen that the AOB abundance in the treatment with vanillic acid is the lowest, significantly lower than the other two inhibitors and the control. Thus, it can be seen that vanillic acid has an obvious inhibitory effect on the abundances of functional microorganisms in nitrification, and can effectively hinder the process of oxidizing ammonium nitrogen (NH4 + ) to nitrite (NO2 - ), achieving the purpose of inhibiting soil nitrification.

[0052] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. Use of vanillic acid in inhibiting soil nitrification.

2. A soil nitrification inhibitor, characterized in that, The soil nitrification inhibitor is vanillic acid.

3. Application of the soil nitrification inhibitor according to claim 2 in inhibiting soil nitrification in crop cultivation.

4. The application according to claim 3, wherein Vanillic acid and nitrogen fertilizer are formulated into a mixed solution and applied to the soil.

5. The application according to claim 4, wherein 100 - 2000 mg of the vanillic acid is applied per kilogram of soil; the application rate of the nitrogen fertilizer is calculated as 100 mg N applied per kilogram of soil.

6. The application according to claim 5, characterized in that 100 - 500 mg of the vanillic acid is applied per kilogram of soil.

7. The application according to claim 6, characterized in that, 100 mg of the vanillic acid is applied per kilogram of soil.

8. The application according to claim 3, wherein The crop cultivation is rice - wheat rotation.

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